Sexual differentiation and the development of secondary sexual characteristics are controlled by different mechanisms across evolution. In vertebrates and some invertebrate groups, these processes are under the control of sex hormones. Given the recent confirmation that insects probably have no sex hormones, the agents responsible for the sexual maturation of arthropods remain under debate. Crustaceans that are evolutionary close to insects possess an androgenic gland (AG) which is responsible for male sexual differentiation.
The role of the AG in male sexual differentiation was demonstrated in several crustacean species by observing primary and secondary sex characteristics after AG removal or transplantation. In the amphipod Orchestia gamarella, bilateral AG ablation diminished spermatogenesis and obstructed the development of secondary male characteristics. In the crayfish Procambarus clarkii, injection of AG extracts protruded external male characteristics. In Macrobrachium rosenbergii, a fully functional sex reversal from males to neo-females and from females to neo-males was achieved by bilateral AG ablation and transplantation, respectively.
The first AG hormone and its two closely related orthologs were found in isopods to be members of the insulin family of hormones as they possess B and A chains with a conserved cysteine residues skeleton, separated by a C peptide present in the pre-hormone and cleaved to give rise to the mature hormone (Ohira et al., Zoolog. Sci. 20: 75-81, 2003).
The insulin-like family of peptides is diverse and widespread. Many multi-cellular organisms were shown to differentially express several different insulin-like peptides. The insulin-like peptides discovered in invertebrates are not confined to glucose metabolism and have roles in metabolism, growth and reproduction. The silkworm Bombyx mori has three insulin-like prothoracicotropic hormones expressed in its brain (Bombyxin I, II and III) regulating ecdysteroids level (Ebbernick et al., Biol. Bull. 177: 176-182, 1989). The freshwater snail Lymnaea stagnalis retains seven insulin-like growth factors expressed in its brain and digestive system, functioning in shell and body growth as well as energy metabolism (Smit et al., Neurosci. 70: 589-596, 1996). In the nematode Caenorhabditis elegans ten insulin-like peptides were divided into three distinct families. Insulin-like peptides from two of these families probably comprise an additional disulfide bond (Duret et al. Genome Res. 8: 348-353, 1998), as is suggested to be the case for the three isopod insulin-like AG factors. Most insulin-like genes, not including insulin-like growth factors, encode a single pre-pro-peptide with a signal peptide and contiguous B, C, and A peptides. The pro-peptide is processed into an active form by linkage of the A and B peptides by disulfide bridges followed by proteolytic cleavage of the C peptide (Riehle et al., Peptides 27: 2547-2560, 2006).
Since the AG has an enormous effect on primary and secondary male characteristics in decapod crustaceans, it is considered not only as a male sex differentiation regulating organ but also as an organ responsible for maintenance of male morphological features. In M. rosenbergii there are three distinct mature male morphotypes, differing in behavior and growth rate. The larger, dominant blue claw (BC) males actively court and protect the females prior to mating. The orange claw (OC) males demonstrate reduced rate of reproductive activities in the presence of dominant males and the small males (SM) practice a form of sneak mating consistent with their small size and higher mobility. An anatomical study demonstrating high gonado-somatic index in BC and SM supported their more intense reproductive behavior compared with OC males which demonstrate poor reproductive skills and invest more effort in somatic growth. In M. rosenbergii, removal of AG from OC males caused attenuation of their transformation to the BC male morphotype, compared with sham operated OC males. The same procedure in SM arrested the dissected individuals in the OC male morphotype. Sun et al. (Aquaculture Internation. 8: 329-334, 1996) found that AG total polypeptide content increased gradually among the male morphotypes with the highest polypeptide content found in BC males. Sun further hypothesized that two polypeptides with apparent molecular weight of ˜16 and ˜18 kDa might be the AG hormone based on size similarity to the AG hormone found in Isopoda and reported the absence of these polypeptides in sexually inactive OC morphotype and presence in sexually active male morphotypes.
Manor et al. (Gen. Comp. Endocrin. 150:326-336, 2007) reported the identification of an AG-specific gene, termed Cq-IAG, in the decapod red-claw crayfish Cherax quadricarinatus. Cq-IAG was shown to be expressed exclusively in AG and its deduced protein was found to be similar in its cysteine backbone and 3D structure to the insulin-like family members (Manor et al., ibid).
U.S. Pat. No. 6,740,794 to Malecha et al. discloses several unidentified polypeptides with apparent molecular weights of 8, 13, 14, 16, 18, and 23-26 kDa obtained from the AG of male prawn M. rosenbergii. Two of these polypeptides, the 16 and 18 kDa, were presumed to be the androgenic sex hormone (AH). U.S. Pat. No. 6,740,794 further discloses uses of AH in treating genetic female shrimp or prawns to produce neomales which can mate with genotypic females to produce female mono-sex shrimp or prawn progeny. U.S. Pat. No. 6,740,794 teaches the advantage of treating female shrimp or prawn with AH as such treatment produces populations of female shrimps and freshwater prawns (M. rosenbergii) having a significant economic advantage over their male counterparts.
U.S. Patent Application Publication No. 2005/0080032 discloses methods for inducing systemic, non-specific, and/or sequence specific immune responses in invertebrates comprising administering to the invertebrate at least one dsRNA that confers immunity against a pathogen, particularly a virus, or modulates expression of a gene that affects growth, reproduction, and general health. Two genes are specifically studied in U.S. Patent Application Publication No. 2005/0080032, a STAT-like gene and an I-KB-Kinase like gene (IKK); both were selected because of the known role of their orthologs in immune responses. U.S. Patent Application Publication No. 2005/0080032 neither discloses nor suggests silencing of a specific insulin-like peptide in invertebrates.
Several crustacean species including the freshwater giant prawn M. rosenbergii exhibit a bimodal growth pattern in which males exhibit superior growth to females. It was shown that in comparison to all-female M. rosenbergii populations, all-male M. rosenbergii populations produce higher marketable yields, grow faster and thus require shorter periods of time to harvest, and give higher income per unit area. The means to produce male monosex M. rosenbergii populations were found to be inefficient and involve tedious procedures such as manual segregation and microsurgical removal of AG from immature males, both procedures require high technical skills.
There is still a need for efficient, technically improved and economically beneficial methods to produce male monosex populations of decapod crustaceans, particularly of M. rosenbergii. 